1. Technical Field
The invention relates to methods and materials involved in the tissue-specific regulation of nucleic acid expression.
2. Background Information
Two components help determine cell-specific nucleic acid and polypeptide expression: transcription factors and regulatory elements. Transcription factors are polypeptides that bind nucleic acid sequences, whereas regulatory elements are specific nucleic acid sequences, usually located near a gene, that are bound by transcription factors. It is the binding of a transcription factor to a regulatory element that typically stimulates transcription of a nearby gene. Thus, the transcription factors available within a cell determine which nucleic acids and polypeptides are expressed by that cell. For example, brain cells contain a set of transcription factors that are unique to brain cells. This set of transcription factors stimulates transcription of brain-specific polypeptides since genes that encode brain-specific polypeptides have nearby regulatory elements that are recognized by transcription factors present within brain cells. Non-brain-specific genes, however, do not have nearby regulatory elements that could be recognized by transcription factors present within brain cells and thus are not expressed. Likewise, non-brain cells do not contain the same set of transcription factors and thus do not express brain-specific nucleic acid and polypeptides.
In eukaryotes, transcription factors have a limited, if not totally cell-type specific, distribution. In addition, each transcription factor typically binds a regulatory element having a very specific nucleic acid sequence. Transcription factors also can function in conjunction with other transcription factors such that the binding to a particular regulatory element is influenced. Thus, the presence of these regulatory elements provides a means for regulating gene expression. Generally, regulatory elements located near transcriptional start sites are known as promoters whereas those located at greater distances from transcriptional start sites are known as enhancers. Further, promoters are typically upstream of the transcriptional start site and only function in one orientation. For example, inverting a promoter sequence usually results in a dramatic decrease in the expression of a downstream gene. Enhancers, however, can be upstream, downstream, or within the nearby gene and can often function in both orientations. For example, an enhancer sequence can be within an intron of the gene it controls.
Many polypeptides and nucleic acids have specific and unique expression patterns. For example, an early polypeptide marker for most, but not all, stem cells in the central nervous system (CNS) is nestin, an intermediate filament polypeptide (Dahlstrand et al., 1995; Frederiksen and McKay, 1988; Hockfield S and R D G McKay, J. Neurosci. 5:3310-3328 (1985); Lendahl et al., 1990; Reynolds B A and S Weiss, Science 255:1707-1710 (1992);Williams B P and J Price, Neuron 14:1-20 (1995)). Upon neuronal differentiation, a switch in intermediate filament gene expression replaces nestin with the neurofilaments (Lendahl U et al., Cell 60:585-95 (1990)); reviewed in Lee M K and D W Cleveland., Annu. Rev. Neurosci. 19:187-217 (1996)). In the adult CNS, nestin is re-expressed in reactive astrocytes (Clarke et al., 1995; Frisen J et al., J. Cell Biol. 131:453-464 (1995)) and in CNS neuroepithelial tumors, most notably gliomas and glioblastomas (Dahlstrand J et al., Cancer Res. 52:5334-5341 (1992) and Tohyama T et al., Lab. Invest. 66:303-313 (1992)). During embryonic development, nestin expression is not restricted to the CNS. Nestin is also found in myogenic precursors (Kachinsky A M et al., Dev. Biol. 165:216-228 (1994); Lendahl U et al., Cell 60:585-595 (1990); Sejersen T and U Lendahl, J. Cell Sci. 106:1291-1300 (1993)), the peripheral nervous system (Dahlstrand J, et al., Dev. Brain Res. 84:109-129 (1995); Hockfield S and RDG McKay, J. Neurosci. 5:3310-3328 (1985); and Stemple D L and D J Anderson, Cell 71:973-985 (1992)), the heart (Kachinsky A M et al., J. Histochem. Cytochem. 43:843-847 (1995)), the developing tooth bud (Terling C et al., Int. J. Dev. Biol. 39:947-956 (1995)) and the testis (Frojdman K et al., Differentiation 61:243-249 (1997)).
Recently, the regulation of nestin gene expression during development was studied using transgenic mice (Zimmerman L B et al., Neuron 12:11-24 (1994)). Interestingly, the upstream promoter region of the gene does not possess any enhancer elements specific for neural stem cells. Instead, two separate enhancer regulatory elements were identified within the rat nestin gene: a myogenic precursor cell-specific enhancer within the first intron and a CNS cell-specific enhancer within the second intron. These results demonstrate the presence of multiple regulatory elements within a single gene that each control its expression in distinct tissues.
Tissue-specific regulatory elements are powerful tools that can direct heterologous nucleic acid and polypeptide expression. Indeed, the second intron from the nestin gene directs heterologous gene expression to the developing nervous system (Lardelli M et al., Mech. Dev. 59:177-190 (1996); Ringstedt T et al., Developnzent 124:2603-2613 (1997); and Lothian C and U Lendahl, Eur. J. Neurosci. 9:452-462 (1997)). Most, if not all, gene therapy approaches benefit from tissue-specific regulatory elements since these elements direct expression of the nucleic acid and/or polypeptide of interest to specific target tissues.